Means and methods for treating angiogenesis-related diseases

This application is a Divisional of U.S. patent application Ser. No. 14/240,273, filed Jun. 24, 2014, which is the U.S. National Phase of International Patent Application No. PCT/EP2012/066467, filed Aug. 23, 2012, which claims priority from U.S. Provisional Patent Application No. 61/526,535, filed Aug. 23, 2011, and European Patent Application No. 11178509.3, filed Aug. 23, 2011. The contents of these applications are incorporated herein by reference in their entirety.

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The present invention is concerned with a protein oligomer comprising at least two NC-1 monomers of human collagen 18 or fragments of an NC-1 monomer of human collagen 18 for use in the treatment or prevention of an angiogenesis-related disease. The invention further pertains to a fusion protein comprising a NC-1 monomer of human collagen 18 and a Fc domain of an immunoglobulin. The invention also relates to a fusion protein comprising: a) an endostatin peptide or endostatin-derived peptide and b) the RGD motif and/or PHSRN motif (SEQ ID NO: 20) of Fibronectin. The invention further relates to a kit comprising the protein oligomer or fusion proteins of the invention.

Endostatin, a 183-amino acid proteolytic cleavage fragment corresponding to the C-terminus of collagen 18 (or collagen XVIII), has been the subject of investigation by a number of laboratories because of its anti-tumor activity and its potential application as an anti-angiogenic cancer therapeutic (O'Reilly et al., 199788, 277; Folkman et al., 2006312, 594; Bergers et al., 1999284, 808). The anti-tumor activity of endostatin is well established. Although the number of separate mechanisms for endostatin action has been proposed, a general consensus on its mechanism is yet absent.

Clinical trials of human endostatin in phase I and II used a recombinant molecule that was expressed in yeast. This formulation of endostatin carried two major handicaps. The half-life of the protein in circulation was very short and 50% of the injected recombinant human endostatin used in the original clinical trials lacked four amino acids at the N-terminus, including two histidines crucial for zinc binding, hence an inactive molecule (Lee et al., 200814, 1487; Boehm et al., 1998252, 190; Tjin et al., 2005, Cancer Res 65, 3656; Sim et al., 1999, Angiogenesis 3, 41). To overcome these deficiencies, a novel recombinant human endostatin expressed and purified inwith an additional nine-amino acid sequence and forming another his-tag structure, called Endostar, was approved by the SDFA in 2005 for the treatment of non-small-cell lung cancer. Endostar suppressed the VEGF-stimulated proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro and blocked microvessel sprouting from rat aortic rings in vitro. Moreover, it could inhibit the formation of new capillaries from pre-existing vessels in the chicken chorioallantoic membrane (CAM) assay and affect the growth of vessels in tumor. It has further been found that the antiangiogenic effects of endostar were correlated with the VEGF-triggered signaling (Ling et al.361, 79). In another study, endostatin fused to the Fc domain of an IgG antibody has been constructed (Lee et al., 200814, 1487). The presence of Fc increased the half-life to longer than a week, analogous to the two angiogenesis inhibitors bevacizumab (Avastin) and VEGF-Trap (Gordon et al., 200119, 843; Holash et al., 200299, 11393).

Although numerous clinical trials proved that endostatin is a very safe drug in a variety of dose schedules, the results did not demonstrate substantial endostatin anti-tumor activity. The dose and schedules may have been sub-optimal, and/or bulky disease in late stage patients may not be optimally responsive to recombinant human endostatin. Therefore, in current clinical trials in China, endostatin is mainly used in combination with chemotherapeutics in order to improve anti-tumor activity of endostatin. For example, in one study, 45 patients with solid tumors were enrolled. All received Endostar at a dose of 7.5 mg/m/day as an intravenous infusion for more than 7 days, in combination with chemotherapy, from 2006 to 2008. No treatment related death occurred in this study. Main reported toxicities included myelosuppression, hepatic impairment, anorexia, nausea, vomiting, diarrhea, febrile and fatigue. No complete response was observed. Two of 42 patients had partial response, twenty-one remained stable, and nineteen had progressive disease. Median time to tumor progression was 3.0 months. Median overall survival was 30.0 months and one year survival rate was 81.0%. This data showed that toxicity of Endostar combined with chemotherapy in the treatment of solid tumors was tolerable with moderate efficacy (Li et al. 201011, 1119-23).

Anti-angiogenic gene therapy has been proposed as an alternative way to continuously provide high concentrations of the anti-angiogenic factors. Gene transfection of anti-angiogenic agents using a viral vector can inhibit the growth of tumors in several mouse models. Viral vectors, however, may cause inflammation and immunological response on repeated injection, and toxicity/safety considerations may preclude the use in humans in the near future. In addition, use of gene-transduced hematopoietic stem cells has been ineffective in an animal model, despite sustained production of endostatin. Furthermore, dosing of biological products using gene vectors is very difficult to standardize due to variation in vector titer, transduction efficiency and expression levels.

There is, thus, a need in the art for improved therapies of angiogenesis-related diseases.

The technical problem underlying the present invention could be seen as the provision of means and methods which comply with the afore-mentioned needs. This technical problem has been solved by the embodiments characterized in the claims and herein below.

Accordingly, the present invention relates to a protein oligomer comprising at least two NC-1 monomers of human collagen 18 or fragments of an NC-1 monomer of human collagen 18 for the treatment or prevention of an angiogenesis-related disease.

The term “collagen 18” and “collagen XVIII” as used herein are used interchangeably and refer to the same protein. The cloning of the mouse and human collagen 18 proteins has been described by Oh et al. (PNAS 1994, 91, 4229; Genomics 1994, 19, 494). The Type XVIII collagen belongs to a unique and novel subclass of the collagen superfamily for which the name “MULTIPLEXIN family” has been proposed. The nucleotide and amino acid sequences of mouse collagen 18 are shown in accession number NM_001109991.1 whereas the corresponding human sequences are shown in NM_030582.3. Further, the amino acid sequences of mouse and human collagen 18 are shown in SEQ ID NOs: 1 and 2, respectively. More specifically, collagen 18 consists of a central, interrupted triple-helical domain flanked at the N-terminus (NC-11 domain) and C-terminus (NC-1 domain) by larger non-triple helical, globular structures (Oh et al., loc. cit.; Abe et al. 1993196, 576).

The C-terminal NC-1 domain (or briefly NC-1) of collagen 18 includes an N-terminal association region (of about 50 amino acid residues), a central protease-sensitive hinge region (of about 70 amino acid residues) and a C-terminal stable endostatin domain (of about 180 amino acid residues) (Sasaki et al., 1998, EMBO J 17, 4249). The endostatin domain comprises a zinc binding site which mediates binding to zinc and is located at the N terminus of endostatin (Ding et al., 1998, PNAS 95, 10443; U.S. Pat. No. 7,524,811). Interestingly, this zinc binding site has been shown to be responsible for the anti-tumor/anti-angiogenic activity of endostatin (Boehm et al., 1998252, 190). The amino acid sequence of the NC-1 domain of the mouse collagen 18 is depicted in SEQ ID NO: 3, whereas the corresponding sequence of the NC-1 domain of human collagen 18 sequence is shown in SEQ ID NO: 4. The association domain of the human NC-1 domain comprising amino acid residues from about 10 to about 60 of the amino acid sequence shown in SEQ ID NO: 4 is responsible for non-covalent trimerization of the NC-1 monomer to form a globular trimer. The proteolytic cleavage-sensitive hinge region comprises amino acid residues from about 61 to about 129 of the amino acid sequence shown in SEQ ID NO: 4. The compact endostatin domain comprises amino acid residues from about 130 to about 308 of the amino acid sequence shown in SEQ ID NO: 4; see, e.g., Sasaki, loc. cit.; Kuo 2001152, 1233; Tjin et al. 200565, 3656. The association region and the endostatin domain in the NC-1 domain are connected by the hinge region (see Sasaki et al., loc. cit.). The hinge region has been found to be cleaved, for instance, by matrix metalloproteinases (MMPs), such as MMP-3, -7, -9, -13 and -20 (Heliasvaara et al.,2005, 307, 192). The above-indicated domain structure of NC-1 is based on structural data. The term “about” as used for the positioning of the domains within NC-1 reflects the fact that the exact boundaries between the mentioned domains may differ from the indicated positions by one, two, three or even more amino acid residues. The exact boundary between, for example, the association domain and the hinge region can be determined, for example, by generating an association domain comprising amino acid residues from about 10 to about 60 of SEQ ID NO: 4 as a starting point and producing shorter fragments thereof, e.g. with a length of 49, 48, 47, 46, 45 and so on, amino acid residues. Said shorter fragments can then be analyzed for their oligomerization properties, i.e. whether they are still able to form oligomers, such as trimers, as the complete association domain does. Alternatively, the endostatin domain may serve as a starting point to address the oligomerization properties of the domains of NC-1. As indicated elsewhere herein, the invention provides for a further method for identifying the exact boundaries of the monomer, dimer and/or trimer transitions in the NC-1 domain as defined herein. However, the above-mentioned domain model fits the gene structure remarkably well, with exons 38 and 39 encoding the association domain, exon 40 the hinge region, and three more exons the endostatin domain (Sasaki et al., loc. cit.).

The term “protein” or “polypeptide” or “peptide” as used herein encompasses isolated or essentially purified (poly)peptides being essentially free of other host cell polypeptides. The term “peptide” as used herein comprises at least two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40 or even more amino acid residues where the alpha carboxyl group of one is bound to the alpha amino group of another. The term “peptide” as used herein encompasses peptidomimetics. As known in the art, peptidomimetics are compounds whose essential elements (pharmacophore) mimic a natural peptide or protein in 3D space and which retain the ability to interact with the biological target and produce the same biological effect; see, e.g., the review by Vagner et al. 2008, Current Opinion in Chemical Biology 12, Pages 292-296. Peptidomimetics are designed to circumvent some of the problems associated with a natural peptide: e.g. stability against proteolysis (duration of activity) and poor bioavailability. Certain other properties, such as receptor selectivity or potency, often can be substantially improved. According to the present invention, the protein or peptide is in one aspect an oligomer. In another aspect, the protein or peptide is a fusion protein, as further defined below. An “oligomer” as used herein means a molecule that comprises a few monomer units, in contrast to a polymer that, at least in principle, comprises an unlimited number of monomers. Preferably, the oligomer is a protein oligomer, i.e. the oligomer comprises two, three, four, five or even more protein monomers, i.e. the oligomer can be, e.g., a dimer, trimer, tetramer, pentamer and so on. A dimer is per definition a macromolecular complex formed by two, usually non-covalently bound, molecules like proteins or peptides. Such a complex can also be formed by protein domains which are parts of protein sequences and structures that can evolve, function, and exist independently of the rest of the protein chains. A homo-dimer is formed by two identical molecules, the underlying process is called homo-dimerization. A hetero-dimer is built by two different macromolecules which are formed by hetero-dimerization. As known in the art, most dimers in biochemistry are not connected by covalent bonds, with the exception of disulfide bridges. Some proteins contain specialized domains to ensure dimerization (or oligomerization), so called dimerization (or oligomerization) domains, as further defined herein below and well known in the art. A trimer is a macromolecular complex formed by three, usually non-covalently bound proteins or protein domains. A homo-trimer is formed by three identical molecules, whereas a hetero-trimer is built by three different molecules. For example, collagen 18 is a homo-trimeric protein. A tetramer consists of four molecules, a pentamer of five molecules, and so on. In these cases, complex formation is often mediated by oligomerization domains, as set forth above. For instance, dimerization can be mediated by an Fc domain of an immunoglobulin or by disulfide bridges as described elsewhere herein, whereas for the trimerization of NC-1 of collagen 18, the association region within the NC-1 domain can be used.

The protein or peptide oligomer of the present invention comprises at least two NC-1 monomers of collagen 18, as defined herein. However, the protein oligomer can comprise also three, four, five, six or even more of said NC-1 monomers of collagen 18, preferably, of human collagen 18. It is also encompassed by the scope of the present invention in some aspects that the protein oligomers or fusion proteins as referred to herein can oligomerize via one or more disulfide bonds. It is further envisaged, that the NC-1 monomers as defined herein are linked covalently, for instance, by chemical cross linking or other methods known in the art.

The term “protein” or “peptide” as used herein includes also protein preparations comprising the protein oligomer or peptide oligomer or fusion protein of the present invention and other proteins in addition. Moreover, the term includes, in an aspect, chemically modified protein or peptide oligomers or fusion proteins. Such modifications may be artificial modifications or naturally occurring modifications.

The protein oligomer or peptide oligomer or fusion protein of the present invention shall have the biological properties referred to herein, preferably anti-angiogenic activities. Such anti-angiogenic activities include, for example, any biological activity inhibiting the growth or migration of endothelial cells and/or pericytes, formation of tubes or endothelium, growth of new capillary blood vessels in the body, slowing or inhibiting of the growth of benign or malignant tumors by cutting off their blood supply, reduce side-effects/toxicity of other anti-tumor or anti-angiogenic agents, e.g., VEGF-Inhibitors, by interference with their mechanism of action, i.e. reduce blood pressure, modulation of inflammatory response in malignant and benign diseases, or improving the patho-physiological parameters, such as perfusion or hypoxia within a therapeutic time window after treatment that, in turn, may facilitate the efficacy of additional therapies (e.g., radiotherapy, chemotherapy or antiapoptotic therapy). The anti-angiogenic activity can be tested by in vitro assays or in vivo by animal models known in the art (Abdollahi et al.,2003, 63, 88902004, 13, 649; PNAS 2007, 104, 128902005, 8, 59; Bergers et al.,1999, 284, 808; Javaherian et al.,2002, 277, 45211; Lee et al.,2008). For instance, the anti-angiogenic activity can be tested in vitro by inhibition of the proliferation and/or migration of endothelial cells stimulated by a growth factor, e.g., by VEGF. In vivo, anti-angiogenic activity can be analyzed, for example, by a chicken chorioallantoic membrane (CAM) assay, whereas an anti-tumor activity can be tested in animal tumor models including, e.g., A549, LLC or H460 non-small cell lung carcinoma, HT29 colon carcinoma, BxPC3 Pancreatic Carcinoma, Karpas 299 lymphoma, MOLM-13 AML (acute myeloid leukemia), 786-0, A2058 cell line (melanoma) or RENCA renal cell carcinoma (RCC) and many others (Abdollahi et al.,. Update 2005, loc. cit.).

The protein oligomer or peptide oligomer or fusion protein of the invention, in an aspect, can be manufactured by chemical synthesis or recombinant molecular biology techniques well known to the person skilled in the art; see, e.g., Sambrook et al. 2001, Molecular cloning: a laboratory manual/Sambrook, Joseph; Russell, David W.—. 3rd ed.—New York: Cold Spring Harbor Laboratory, 2001. In an aspect, such a method of manufacturing the protein oligomer or peptide oligomer or fusion protein of the present invention comprises (a) culturing a host cell comprising a nucleic acid encoding the protein oligomer or peptide oligomer or fusion protein of the invention and (b) obtaining from the host cell of step (a) the protein oligomer or peptide oligomer or fusion protein, and, optionally, storing the protein oligomer or peptide oligomer or fusion protein. Preferably, said method is carried out under serum-free conditions, since it has been found by the present inventors that protein oligomers comprising two or more NC-1 monomers as defined herein are susceptible to degradation in serum or cell culture medium comprising serum. In an aspect of this method, the protein oligomer or peptide oligomer or fusion protein can be obtained by conventional purification techniques from, e.g., a host cell lysate including, but not limited to, affinity chromatography, ion exchange chromatography, size exclusion chromatography and/or preparative gel electrophoresis.

In one embodiment of the protein oligomer or peptide oligomer or fusion protein of the invention, the “NC-1 monomer”, “NC-1 monomer of collagen 18” or “NC-1 monomer of human collagen 18” as used herein in the protein oligomer or peptide oligomer or fusion protein of the invention comprises at least one part, i.e. at least one domain, region or fragment, of the non-collagenous NC-1 domain of human collagen 18, as defined herein. It is preferred that the NC-1 monomer is human. The NC-1 monomer as used herein comprises, in one aspect of the protein oligomer or peptide oligomer or fusion protein of the invention, at least one endostatin-derived peptide or endostatin peptide, comprising the zinc binding site/domain of the endostatin domain. The human endostatin zinc binding site is formed by histidines 1, 3 and 11 and aspartic acid 76 (Ding et al., loc. cit.). It has been reported that zinc binding of endostatin is essential for its anti-angiogenic activity (Boehm et al., loc. cit.). Further, Tjin et al. (loc. cit.) found that a 27 amino amino acid synthetic peptide corresponding to the N-terminal zinc binding domain of endostatin is responsible for its antitumor activity. The term “endostatin peptide” as used herein means that the amino acid sequence of this peptide can be found in the endostatin domain of NC-1. The term “endostatin-derived peptide” means that such a peptide can differ from the corresponding endostatin peptide in the endostatin domain of NC-1, in one, two, three, four or even more amino acid residues, while at least maintaining (or even exceeding) the biological activity (as described elsewhere herein) of the corresponding endostatin peptide in the endostatin domain of NC-1. Examples of endostatin peptides comprising said zinc binding site/domain of the endostatin domain and exhibiting anti-angiogenic and/or anti-tumor activity have been described, e.g., in Tjin et al., loc. cit., or in U.S. Pat. No. 7,524,811. Preferably, the endostatin-derived peptide or endostatin peptide is about 10 to about 40 amino acid residues in length, preferably 23 to 35, more preferably 24, 25, 26, 27, 28, 29 or 30 amino acid residues. For example, SEQ ID NO: 9 shows the corresponding murine sequence of the active motif of NC-1-endostatin domain (ED) (i.e., the amino-terminal zinc binding domain mediating antiangiogenic and/or antitumor activity) with a length of 26 amino acid residues, whereas SEQ ID NO: 10 shows the corresponding human sequence with a length of 25 amino acid residues. The Histidines in these sequences are particularly important since it has been found by the present inventors in a previous study, that substitution of said Histidines by Alanine residues abolished antitumor and antiangiogenic activity; see Example 2.10. It is within the scope of the present invention that said NC-1 monomer comprises more than one endostatin-derived peptide or endostatin peptide, for example, two, three, four or even more peptides.

In preferred embodiments of the protein oligomer or peptide oligomer or fusion protein of the invention, the NC-1 monomer of the invention comprises or consists of the endostatin domain, as defined elsewhere herein. Preferably, the mentioned endostatin-derived peptide, endostatin peptide or endostatin domain carry a single mutation of glutamine to cysteine at position 7 of the endostatin domain. Such mutants are able to form disulfide bridges and are, thus, able to form dimers; see, e.g., Kuo 2001152, 1233; Tjin et al. 200565, 3656.

In a further embodiment, the NC-1 monomer of the invention comprises, in addition to the zinc binding site/domain of the endostatin domain, the endostatin-derived peptide, the endostatin peptide or the endostatin domain, a hinge region. Such a construct will probably form a monomer, possibly a dimer. The formation of a dimer cannot be excluded since it appears that the hinge region may also contribute to the dimer association of such constructs. Optionally, such an NC-1 monomer comprises, in addition, to the mentioned domain constituents an association domain, i.e. the non-triple helical trimerization domain of human collagen 18, or another oligomerization domain as referred to herein. It is evident to those skilled in the art that the presence of the association domain results in the formation of a trimer. In another aspect, the NC-1 monomer comprises an endostatin domain and an association domain of the above-defined NC-1 domain and, in a still further aspect, an association domain, a hinge region and an endostatin domain, each of said NC-1 domain. In the latter aspect, the NC-1 monomer comprises the complete NC-1 domain of human collagen 18 or is, i.e. consists of, the NC-1 domain of human collagen 18 (of about 38 kDa). The NC-1 domain of human collagen 18 and the structure of said NC-1 domain has been defined, e.g., by Sasaki et al. (loc. cit.). The NC-1 domain of collagen 18 consists of a non-triple-helical sequence of 315 (mouse) or 312 (human) amino acid residues. As set forth above, the NC-1 domain has been found to associate non-covalently to form a trimer via the above-mentioned association domain.

Oligomerization of NC-1 is mediated by at least two domains of this protein: one consisting of approximately 50 amino acids at the N-terminal of the protein defining a triple-helix structure, i.e. the association domain. The second domain which participates in oligomerization is located at the N-terminus of endostatin and is able to bind to zinc. The human endostatin zinc site is formed by histidines 1, 3 and 11 and aspartic acid 76. Said domain has been shown to form a dimer at high concentration of endostatin (Ding et al., loc. cit.). It is also possible that the protease sensitive hinge region plays a role in oligomerization of NC-1, as already indicated above. Accordingly, in some aspects of the invention, the NC-1 monomer can further comprise a hinge region of the NC-1 domain.

The NC-1 monomer of the invention is preferably longer than 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, or 310 amino acid residues. In case the NC-1 monomer comprises the association domain, the hinge region and the zinc binding site/domain of endostatin domain or the complete endostatin domain, it is preferred that the NC-1 monomer is longer than 312 amino acid residues and comprises even more preferred at least 315, 320, 330, 340, 350, 400, 500 or even more amino acid residues.

The term “oligomerization domain” as used herein refers generally to a protein domain which mediates the sub-unit assembly of the two or more NC-1 monomers, as defined herein. As indicated above, the oligomerization domain mediates dimerization, trimerization, and/or tetramerization and so on, of the NC-1 monomers. Such oligomerization leads, e.g., to functional advantages of multivalency and high binding strength, increased structure stabilization and combined functions of different domains, resulting in enhanced biological activity, such as improved or increased anti-angiogenic and/or anti-tumor activity. In one aspect, the oligomerization domain comprises the association domain of the NC-1 domain mentioned above, i.e. the non-triple helical trimerization domain of collagen 18 which is responsible for non-covalent oligomerization of the NC-1 monomers or the collagen 18 helices. In another aspect, the oligomerization domain can comprise other scaffold constructs/domains providing oligomerization and longer half life, well known in the art; see, e.g. Ali and Imperiali 200513, 5013. Such an oligomerization domain replaces structurally and functionally the association domain as found in the natural human NC-1 domain referred to above, or is used, in addition, to said association domain. In a further embodiment of the protein oligomer or fusion protein of the invention, the oligomerization is mediated by an Fc domain of an immunoglobulin, i.e. the oligomerization domain of the NC-1 monomer as defined herein comprises or is a Fc domain of an immunoglobulin. It is known in the art that fusion of a Fc domain to, e.g., a peptide or protein mediates a longer half life in circulation. It is to be understood that the Fc domain may be used in said NC-1 monomer, in addition, to the association domain of the NC-1 domain mentioned above (as shown, for instance in the following examples) or may replace the association domain. The Fc domain confers a dimeric structure on the NC-1 monomer as defined herein since Fc is a dimer itself. In another embodiment, the oligomerization can be mediated by the introduction of a structural modification, e.g., a mutation into the NC-1 monomer which results in the formation of disulfide bonds, as set forth in more detail below. It is further envisaged that the protein oligomers or peptide oligomers or fusion proteins of the invention can be formed covalently.

The invention further relates to a method for identifying the exact boundaries of the monomer, dimer and/or trimer transitions in the NC-1 domain as defined herein, the method comprising: a) generating a series of recombinant peptides from or derived from the NC-1 domain, starting with a peptide consisting of the endostatin domain, followed by increasing the size of said peptide consisting of the endostatin domain in steps of about 10 to 20 amino acid residues, and b) testing the recombinant peptides of step a) for their oligomerization properties, i.e. whether said peptides are able to form dimers or trimers and identifying peptides which are able to form oligomers, and c) determining the exact boundaries of the monomer, dimer and/or trimer transitions in the NC-1 domain. The method can comprise a further step d) of constructing an oligomer or fusion protein of the invention using the recombinant peptides identified in step b) which are able to form dimers or trimers. For generating a series of recombinant peptides from or derived from the NC-1 domain, peptide or protein synthesis known in the art can be used. The term “derived from” has been defined elsewhere herein and appliesto peptides derived from the NC-1 domain. For testing the oligomerization properties of said fragments, Western blot analysis, immunoprecipitation, SDS-PAGE, chromatographic methods or other methods well known in the art can be utilized. The recombinant peptides generated by the above-indicated method can be used to produce oligomers or fusion proteins, such as Fc fusion proteins, of the invention which can then further be tested for their anti-angiogenic and/or anti-tumor activity. The invention further pertains to the recombinant peptides from or derived from the NC-1 domain identified by such a method which show the biological activity as defined elsewhere herein, preferably anti-angiogenic and/or anti-tumor activity. An oligomer or fusion protein of the invention comprising such peptides is particularly useful as a pharmaceutical composition, as set forth elsewhere herein. The invention also relates to recombinant peptides from or derived from the NC-1 domain which are generated by increasing the size of the endostatin domain in steps of about 10 to 20 amino acid residues. Each of these peptides is a candidate for exploring their anti-angiogenic and/or anti-tumor activity by using in vitro and/or in vivo assays described elsewhere herein.

The “NC-1 monomer” of human collagen 18 as defined herein can comprise additional protein domains or subunits, for instance, the above-mentioned Fc domains of immunoglobulins, or protein tags, for example, His tags or the like, which can be used, e.g., for purification and/or detection. As well known in the art, protein tags are peptide sequences genetically grafted onto a recombinant protein. These tags can in one aspect be removable by chemical agents or by enzymatic means, such as proteolysis or intein splicing. Such tags are attached to the NC-1 monomer as referred to herein. Affinity tags are appended to proteins so that they can be purified from their crude biological source such as a cell lysate using an affinity technique well known in the art. These include, for example, chitin binding protein (CBP), maltose binding protein (MBP), Fc domains of immunoglobulins or glutathione-S-transferase (GST). The poly(His) tag is a widely-used protein tag; it binds to metal matrices. Solubilization tags are used, especially for recombinant proteins expressed in chaperone-deficient species such as, to assist in the proper folding in proteins and keep them from precipitating. These include, e.g., thioredoxin (TRX) and poly-(NANP). Some affinity tags have a dual role as a solubilization agent, such as MBP, and GST. Chromatography tags are used to alter chromatographic properties of the NC-1 monomer to afford different resolution across a particular separation technique. Often, these consist of poly-anionic amino acids, such as the FLAG-tag. Epitope tags are short peptide sequences which are chosen because high-affinity antibodies can be reliably produced in many different species. These are usually derived from viral genes, which explain their high immunoreactivity. Epitope tags include, for instance, V5-tag, c-myc-tag, and HA-tag. These tags are useful, e.g., for western blotting and immunoprecipitation experiments, although they also find use in protein purification. Fluorescence tags are used to give visual readout on a protein. GFP and its variants are the most commonly used fluorescence tags. More advanced applications of GFP include using it as a folding reporter (fluorescent if folded, colorless if not). Protein tags find many other usages, such as specific enzymatic modification (such as biotin ligase tags) and chemical modification (Flash tag). The various tags can also be combined to produce multifunctional modifications of the NC-1 monomer. The NC-1 monomer of human collagen 18 as defined herein can also comprise radioisotopes, e.g.III, Cu-64, Cu-67, Y-86, Zr-89, Y-90, Re-188, Ga-68; or radionuclides binding to chelates such as DTPA; toxins, e.g. Diphtheria toxin, or apoptosis inducing agents; or chemicals, e.g. chemotherapies such as taxols, or gemcitabine, which may be useful in improving and/or detecting the anti-angiogenic activity of the protein oligomer or fusion protein of the invention. In other embodiments, the protein oligomer or fusion protein of the invention is pegylated. Pegylation is the process of covalent attachment of polyethylene glycol (PEG) polymer chains to another molecule, normally a drug or therapeutic protein. Pegylation is routinely achieved by incubation of a reactive derivative of PEG with the target macromolecule. The covalent attachment of PEG to a drug or therapeutic protein can “mask” the agent from the host's immune system (reduced immunogenicity and antigenicity), increase the hydrodynamic size (size in solution) of the agent which prolongs its circulatory time by reducing renal clearance. Pegylation can also provide water solubility to hydrophobic drugs and proteins. Pegylation of compounds is well known in the art; see, e.g., Damodaran and Fee 201015, 18.

The term “Fc region” or “Fc domain” as used herein means the fragment crystallizable region which is the tail region of an antibody or immunoblobulin that interacts with cell surface receptors, i.e. Fc receptors, and some proteins of the complement system. This property allows antibodies to activate the immune system. In IgG, IgA and IgD antibody isotypes, the Fc domain is composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains; IgM and IgE Fc domains contain three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. The Fc domains of IgGs bear a highly conserved N-glycosylation site. Glycosylation of the Fc fragment is essential for Fc receptor-mediated activity. The N-glycans attached to this site are predominantly core-fucosylated diantennary structures of the complex type. In addition, small amounts of these N-glycans also bear bisecting GlcNAc and α-2,6 linked sialic acid residues. Fusion of the Fc domain of immunoglobulins to proteins has been found to enhance the production and secretion of the fusion proteins in mammalian cells (Lo et al., 199811, 495, Capon et al., 1989337, 525). In addition, linking of angiogenesis inhibitors to an immunoglobulin Fc domain have shown to increase the half life of said inhibitors (Capon et al. 1989337, 525; Gordon et al., 200119, 843; Holash et al., 200299, 11393). However, the Fc domain can not only be used for purification, solubilization and/or detection purposes but alters advantageously the biological properties of the protein oligomer or fusion protein of the invention, as set forth herein below and in the following examples. In one embodiment, the one or more Fc domains can be cleaved off by treatment with proteases, such as enterokinase or thrombin, if desired. Preferably, the Fc domain as referred to herein is from human IgG (Bergers and Javaherian1999; Lee et al2008). As evident to those skilled in the art, in principle, any IgG isoform can be used to generate the oligomer or fusion protein of the invention. Even subfragments or single chains of the Fc domain of IgG can be used in order to prolong the half life or oligomerization of the oligomer or fusion protein of the invention. The amino acid sequences of a mouse and human Fc domain which can be used for the generation of an oligomer or a fusion protein of the invention, e.g. an Fc-NC-1 or NC-1-Fc fusion protein, are shown in SEQ ID NOs: 5 and 6, respectively.

The term “angiogenesis-related disease” as used herein denotes any disorder associated with abnormal blood vessel growth, either excessive or insufficient. The term “angiogenesis-related disease” is preferably selected from the group consisting of angiogenesis-dependent cancer including solid tumors, blood born tumors such as leukemias, melanomas, tumor metastases, benign tumors such as hemangiomas, acoustic neuromas, neurofibromas, trachomas, pyogenic granulomas; rheumatoid arthritis; psoriasis; ocular angiogenic diseases such as diabetic reintopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasias, rubeosis; Osler-Webber syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; wound granulation; diseases of excessive or abnormal stimulation of endothelial cells such as interstinal adhesions, atherosclerosis, scleroderma, hypertrophic scars (keloids); diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa) and ulcers (). Preferably, the angiogenesis-related disease as referred to herein is a melanoma.

The term “treatment” as used herein denotes the improvement or even elimination of one or more symptoms associated with the angiogenesis-related disease as referred to herein, by the administration of a protein oligomer or peptide oligomer or fusion protein of the invention. An improvement may also be seen as a slowing or stopping of the progression of the angiogenesis-related disease as set forth herein.

The term “prevention” as used herein means the avoidance of the occurrence or re-occurrence of an angiogenesis-related disease as specified herein, by the administration of a protein oligomer or peptide oligomer or fusion protein of the invention.

It has unexpectedly been found by the present inventors that trimeric NC-1 (with NC-1 comprising the association domain, the hinge region and the endostatin domain) derived from human collagen 18 binds fibronectin, whereas endostatin monomer lacks binding to fibronectin. Fibronectin is recognized as a major extracellular matrix protein, binding angiogenic and anti-angiogenic reagents. Endostatin is a monomer under physiological conditions. The major precursor to endostatin is NC-1, a trimeric molecule consisting of three interlinked chains, each with approximately 330 amino acids. This shows that NC-1 trimer has distinct properties in comparison to endostatin. Furthermore, an Fc-endostatin which forms dimers as well as an artificial endostatin dimer bearing a single mutation in amino acid position 7 (glutamine to cysteine) of endostatin retains binding to fibronectin indicating the importance of oligomerization for binding to fibronectin. Following a search for endostatin-size molecules in human sera, the inventors failed to identify the conventional size endostatin (of about 20 kDa). The appearance of endostatin size molecules in human blood circulation might be due to the degradation of NC-1 trimer by proteases following collection of human sera. NC-1 trimer appeared to be the major physiological product of collagen 18 degradation, present in tissues and circulation showing distinct biological properties not shared by (monomeric) endostatin. The inventors further demonstrated high affinity binding of fibronectin to VEGF, NC-1 trimer as well as co-immunoprecipitated these three candidate interaction partners from peripheral blood platelets protein lysates. Furthermore, in-vivo co-localization of NC-1 trimer, Fibronectin, VEGF and alpha 5 beta 1 (α5β1) integrin could be demonstrated, suggesting a model in which an ensemble of VEGF, NC-1 trimer, integrin α5β1 with fibronectin prelude the initiation of the anti-angiogenic process. Most importantly, antitumor studies of NC-1 trimer versus endostatin showed that NC-1 trimer is a more potent anti-angiogenic protein than endostatin. The above data are specified in more detail in the following examples.

In one embodiment of the protein oligomer of the invention, the NC-1 monomer of human collagen 18 comprises an (i) oligomerization domain, (ii) a hinge region and/or (iii) endostatin domain or a fragment of said endostatin domain and, optionally recombinant protease cleavage site within the hinge region. Preferably, said fragment of the endostatin domain is a peptide comprising the zinc binding site/domain of endostatin.

In another preferred embodiment of the protein oligomer of the invention, the hinge region is interposed between the oligomerization domain and the endostatin domain. Preferably, the hinge region is located between the oligomerization domain and the zinc binding site/domain of endostatin or endostatin domain in the NC-1 monomer as referred to herein. The domain arrangement within the NC-1 monomer of human collagen 18 is preferably oligomerization domain-hinge region-endostatin domain, or endostatin domain-hinge region-oligomerization domain.

Optionally, the hinge region within the NC-1 monomer of human collagen 18 may comprise one or more recombinant protease cleavage sites, in addition to the endogenous protease cleavage sites of the hinge region. Such a recombinant protease cleavage site can be, for instance, an enterokinase or thrombin cleavage (Bergers and Javaherian; Lee et al.; loc. cit.). Cleavage by the respective protease allows for, e.g., the release of the endostatin domain(s) of the protein oligomer or fusion protein of the invention.

In a preferred embodiment of the protein oligomer or peptide oligomer or fusion protein of the invention, the oligomerization domain comprises a non-triple helical trimerization domain of human collagen 18 (i.e. the association domain), an Fe domain and/or an artificial oligomerization domain. The oligomerization domain comprises in one aspect a non-triple helical trimerization domain of human collagen 18 which is responsible for trimerization of the three chains of the NC-1 domain. In another aspect, it comprises an Fe domain. The Fc domain confers a dimeric structure on the NC-1 monomer as defined herein since the Fe domain is a dimer itself. In a third aspect, it comprises an artificial oligomerization domain, for example, cysteins resulting in disulfide bridges between two monomers which replaces structurally and functionally the association domain as found in the natural human NC-1 referred to above, or is used in addition to said association domain. It is also encompassed by the scope of the invention, that the oligomerization domain of the protein oligomer or fusion protein of the invention comprises a non-triple helical trimerization domain of human collagen 18 and a Fc domain. Further, it can comprise an artificial oligomerization domain and a Fe domain.

Preferably, the Fc domain is from IgG or other immunoglobulin isoforms as well as other scaffold constructs providing oligomerization and longer half life described in the art; see, e.g., Lo et al Protein Enginerring 1998, 11, 495. A murine Fc domain is shown, for example, in SEQ ID NO: 5. More preferably, the Fe domain is from a human IgG, even more preferred from human IgG1. Particularly preferred, the human Fe domain comprises or consists of an amino acid sequence as shown in SEQ ID NO: 6.

The oligomerization domain of the NC-1 monomer can be a Fc domain of an immunoglobulin, preferably a Fc domain from IgG1, as set forth above. The protein oligomer or peptide oligomer or fusion protein of the invention can also contain two, three or even more Fc domains. In one aspect, the Fc domain(s) may be cleaved off the protein oligomer or peptide oligomer or fusion protein of the invention, if desired. For instance, an artificial protease cleavage site such as an enterokinase or a thrombin cleavage site can be interposed between the NC-1 monomer and the Fc domain(s) in the protein oligomer or peptide oligomer of the invention, for example, via a corresponding (poly)peptide linker. Upon cleavage by the respective protease, the oligomer is released from the Fc domain(s). The Fc domain(s) can be used for purification and/or detection. In addition, the Fc domain alters the biological properties of the protein oligomer or fusion protein of the invention, such as half-life extension in circulation and improvement of biological activity, preferably improvement of anti-angiogenic activity. For example, it has been found that an Fc-endostatin fusion protein is able to bind fibronectin as a dimer, whereas endostatin monomer does not. Moreover, Fc-endostatin shows a longer half-life than endostatin.

In a further preferred embodiment of the protein oligomer or peptide oligomer or fusion protein of the invention, the artificial oligomerization domain comprises a single mutation at position 7 of the endostatin domain in which glutamine is replaced by cysteine. Preferably, the monomer as defined herein comprises in some aspects a single mutation of glutamine to cysteine at position 7 of the endostatin domain. For example, it has been found that a recombinantly introduced enterokinase cleavage site between the Fc domain and endostatin domain in a fusion protein results in the formation of a dimer upon enterokinase cleavage because of disulfide bond formation between adjacent C7 residues in the endostatin domains; see Kuo 2001152, 1233. As set forth above, NC-1 trimer and endostatin dimers have distinct properties, in comparison to the endostatin monomer. The above mutation at position 7 (glutamine to cysteine) can also be introduced in the N-terminal peptide of endostatin which has been shown to represent the antitumor domain of endostatin (Tjin et al. 200565, 3656). The oligomerization of the peptide can be achieved by either artificial dimerization as described above or simply by recombinant fusion to the Fc moiety without a mutation in position 7. An example for a fusion protein of the invention comprising said mutation at position 7 mediating dimerization is shown in SEQ ID NO: 15; see Example 2.10.

In another preferred embodiment of the protein oligomer or peptide oligomer of the invention, the recombinant protease cleavage site within the hinge region is an enterokinase or thrombin cleavage site. The cleavage of the protein oligomer or peptide oligomer with the enterokinase or thrombin results in the release of the endostatin domains from the protein oligomer or peptide oligomer of the invention.

In a further preferred embodiment of the protein oligomer or peptide oligomer of the invention, the NC-1 monomer as defined herein contains only protease cleavage sites naturally occurring within the hinge region, i.e. it does not comprise a recombinant protease cleavage site. In this case, the hinge region can be cleaved, e.g. by MMPs, as set forth elsewhere herein, in order to release, e.g., the endostatin domain(s). In another aspect, these naturally occurring protease cleavage sites in the hinge region of the NC-1 monomer can be mutated so that NC-1 monomer is no longer cleaved by said proteases. In this way, the anti-angiogenic activity of the protein oligomer of the invention may still be improved.

In another embodiment of the protein oligomer or peptide oligomer of the invention, the angiogenesis-related disease to be treated is selected from the group consisting of angiogenesis-dependent cancer including solid tumors, melanomas, tumor metastases, blood born tumors such as leukemias, benign tumors such as hemangiomas, acoustic neuromas, neurofibromas, trachomas, pyogenic granulomas; rheumatoid arthritis; psoriasis, ocular angiogenic diseases such as diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasias, rubeosis, Osler-Webber syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma, wound granulation, diseases of excessive or abnormal stimulation of endothelial cells such as intestinal adhesions, atherosclerosis, scleroderma, hypertrophic scars (keloids), diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa) and ulcers (). Preferably, the angiogenesis-related diseases are renal cell carcinoma, colorectal-, prostate-, breast- or lung cancer.

The protein oligomer or peptide oligomer or fusion protein of the invention is preferably formulated as a pharmaceutical composition which can be administered by standard routes. Generally, the pharmaceutical composition may be administered by the topical, transdermal, intraperitoneal, intracranial, intracerebroventricular, intracerebral, intravaginal, intrauterine, oral, rectal or parenteral (e.g. intravenous, intraspinal, subcutaneous or intramuscular) route.

A pharmaceutical composition comprising the protein oligomer or peptide oligomer or fusion protein of the invention as pharmaceutical active compound may be used for non-human or preferably human therapy of various angiogenesis-related diseases or disorders as specified elsewhere herein in a therapeutically effective dose. In an aspect, the protein oligomer or peptide oligomer or fusion protein of the invention can be present in liquid or lyophilized form. In an aspect, the protein oligomer or peptide oligomer or fusion protein can be present together with glycerol, protein stabilizers (e.g., human serum albumin (HSA)) or non-protein stabilizers.

The compound (i.e. the protein oligomer or peptide oligomer or fusion protein of the invention) is the active ingredient of the pharmaceutical composition, and is in one aspect, administered in conventional dosage forms prepared by combining the drug with standard pharmaceutical carriers according to conventional procedures. These procedures may involve mixing, granulating, and compression, or dissolving the ingredients as appropriate to the desired preparation. It will be appreciated that the form and character of the pharmaceutical acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration, and other well-known variables.

The carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and being not deleterious to the recipient thereof. The pharmaceutical carrier employed may include a solid, a gel, or a liquid. Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatine, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Exemplary of liquid carriers are phosphate buffered saline solution, syrup, oil, water, emulsions, various types of wetting agents, and the like. Similarly, the carrier or diluent may include time delay material well known to the art, such as glyceryl mono-stearate or glyceryl distearate alone or with a wax. Said suitable carriers comprise those mentioned above and others well known in the art, see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania.

The diluent(s) is/are selected so as not to affect the biological activity, preferably, anti-angiogenic activity of the combination. Examples of such diluents are distilled water, physiological saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers and the like.

A therapeutically effective dose refers to an amount of the protein oligomer or peptide oligomer or fusion protein of the invention to be used in a pharmaceutical composition which prevents, ameliorates or treats the symptoms accompanying an angiogenesis-related disease or condition referred to in this specification. Therapeutic efficacy and toxicity of the compound can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.

The dosage regimen will be determined by the attending physician and other clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Progress can be monitored by periodic assessment.

The medicament referred to herein is administered at least once in order to treat or ameliorate or prevent a disease or condition recited in this specification. However, the said medicament may be administered more than one time.

Specific pharmaceutical compositions are prepared in a manner well known in the pharmaceutical art and comprise at least one active compound referred to herein above in admixture or otherwise associated with a pharmaceutically acceptable carrier or diluent. For making those specific pharmaceutical compositions, the active compound(s) will usually be mixed with a carrier or the diluent. The resulting formulations are to be adapted to the mode of administration. Dosage recommendations shall be indicated in the prescribers or users instructions in order to anticipate dose adjustments depending on the considered recipient.

The pharmaceutical composition may in a further aspect of the invention comprise drugs in addition to the protein oligomer of the invention which are added to the medicament during its formulation. Finally, it is to be understood that the formulation of a pharmaceutical composition takes place under GMP standardized conditions or the like in order to ensure quality, pharmaceutical security, and effectiveness of the medicament.